Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. By binding to a specific component of a cell, a fluorescent dye can be used to detect the presence of that component in the cell and to distinguish cells containing the component. The presence of specific components in a cell can be indicative of certain conditions in the cell.
A family of highly permeant nucleic acid stains was described in the copending parent application CYCLIC-SUBSTITUTED UNSYMMETRICAL CYANINE DYES, Ser. No. 08/090,890 filed Jul. 12, 1993 by Haugland, et al. (incorporated by reference). These fluorescent dyes are similar to the compounds first described Brooker, et al., J. AM. CHEM. SOC. 64, 199 (1942) and lower alkyl (1-6) substituted unsymmetrical cyanine dyes exemplified by Thiazole Orange, as disclosed in U.S. Pat. Nos. 4,883,867 (1989) and 4,957,870 (1990) to Lee, et al. (both incorporated by reference), except that the cyclic-substituted compounds were found to stain the nucleic acids of viable and non-viable bacteria, plant and animal cells more quickly and completely.
A subset of these highly permeant stains was unexpectedly found to cause the formation of distinctive oblong or spherical structures in the vacuoles of fungi, particularly in the vacuoles of yeasts. Formation of these structures in the vacuoles does not appear to be related to nucleic acid staining and has not been described as occurring in other cells. Thus, although these dyes stain the nucleic acids of a wide variety of cells, they only induce the formation of the distinctive intravacuolar structures in fungi, particularly in yeast. The dyes that possess this property are substituted at the 2-position by halogen or other suitable functional groups that can be displaced by nucleophiles. A similarly 2-substituted derivative of Thiazole Orange does not produce these structures, nor do other cyclic-substituted dyes without these specific substituents at the 2-position (even if appropriately substituted at other positions). The dyes of the present invention induce the formation of distinctive structures in metabolically active cells but not in dead cells, making the dyes useful in determining the viability of yeast microorganisms and other fungi.
The dyes of this invention have particular advantages over other dyes that are used to distinguish viable yeast cells. Because the dyes of this invention can also be used to detect bacteria (but without the formation of the distinctive intravacuolar structures), a sample can be screened for viable yeast and contaminating bacteria at the same time. In addition, because the structures are typically fluorescent, their optical properties can be used to automate the detection and counting of cells containing such structures. This is not the case for the colored dye methylene blue, which is the most common dye used to determine yeast viability. Methylene blue staining relies on the reduction of the dye to a colorless form inside the live cell to differentiate living from non-living yeast cells. This mechanism often leads to ambiguous results as the oxidation-reduction potential varies between cells. Furthermore, the evaluation of viable yeast cells is hampered by a high background color, the difficulty in discriminating between similar shades of blue, and the presence of "false deads" in the sample (living yeast cells that nevertheless remain colored when stained with methylene blue).
Fluorescent thioflavin compounds are disclosed in U.S. Pat. No. 4,554,546 (to Wang, et al. 1985) for staining a wide range of nucleic acids, including the nucleic acids of yeast and fungi. These compounds and similar nucleic acid stains disclosed in U.S. Pat. No. 5,057,413 (to Terstappen et al. 1991) and in U.S. Patent No. 4,937,198 (to Lee et at. 1990) differ front the core chemical structure of the compounds used for Applicants' invention and do not contain an appropriate functional group susceptible to nucleophilic displacement. In addition, the thioflavin compounds do not form structures that allow the discrimination between metabolically active and inactive cells.
Fluorescent bodies in the vacuoles of yeast stained with DAPI have been reported, e.g. Tijssen, et al., BIOCHIM. BIOPHYS. ACTA 721, 394 (1982); Allan, et al., CAN. J. MICROBIOL. 26, 912 (1980) but the authors do not correlate the appearance of these bodies with yeast viability, and these dyes also do not possess a functional group susceptible to nucleophilic displacement. Furthermore, although DAPI dyes have high fluorescent enhancements, they require UV excitation and emit a blue fluorescence, making them more difficult to detect over the background cellular autofluorescence.
There is presently no fungal viability stain that combines all of the desirable properties of the present invention. The dyes of the present invention give highly fluorescent staining at wavelengths where cellular autofluorescence is relatively low, with very low background signal, easy interpretation of staining results, and good correlation with cell metabolism. In addition, the dyes of the present invention allow the simultaneous detection of bacterial contamination in a sample containing fungi, as they stain all bacteria indiscriminately.